In vivo functions of mitogen-activated protein kinases: conclusions from knock-in and knock-out mice

Multicellular organisms achieve intercellular communication by means of signalling molecules whose effect on the target cell is mediated by signal transduction pathways. Such pathways relay, amplify and integrate signals to elicit appropriate biological responses. Protein kinases form crucial intermediate components of numerous signalling pathways. One group of protein kinases, the mitogen-activated protein kinases (MAP kinases) are kinases involved in signalling pathways that respond primarily to mitogens and stress stimuli. In vitro studies revealed that the MAP kinases are implicated in several cellular processes, including cell division, differentiation, cell survival/apoptosis, gene expression, motility and metabolism. As such, dysfunction of specific MAP kinases is associated with diseases such as cancer and immunological disorders. However, the genuine in vivo functions of many MAP kinases remain elusive. Genetically modified mouse models deficient in a specific MAP kinase or expressing a constitutive active or a dominant negative variant of a particular MAP kinase offer valuable tools for elucidating the biological role of these protein kinases. In this review, we focus on the current status of MAP kinase knock-in and knock-out mouse models and their phenotypes. Moreover, examples of the application of MAP kinase transgenic mice for validating therapeutic properties of specific MAP kinase inhibitors, and for investigating the role of MAP kinase in pathogen-host interactions will be discussed.     

A conserved Pol? binding module in Ctf18-RFC is required for S-phase checkpoint activation downstream of Mec1

Defects during chromosome replication in eukaryotes activate a signaling pathway called the S-phase checkpoint, which produces a multifaceted response that preserves genome integrity at stalled DNA replication forks. Work with budding yeast showed that the ‘alternative clamp loader’ known as Ctf18-RFC acts by an unknown mechanism to activate the checkpoint kinase Rad53, which then mediates much of the checkpoint response. Here we show that budding yeast Ctf18-RFC associates with DNA polymerase epsilon, via an evolutionarily conserved ‘Pol ϵ binding module’ in Ctf18-RFC that is produced by interaction of the carboxyl terminus of Ctf18 with the Ctf8 and Dcc1 subunits. Mutations at the end of Ctf18 disrupt the integrity of the Pol ϵ binding module and block the S-phase checkpoint pathway, downstream of the Mec1 kinase that is the budding yeast orthologue of mammalian ATR. Similar defects in checkpoint activation are produced by mutations that displace Pol ϵ from the replisome. These findings indicate that the association of Ctf18-RFC with Pol ϵ at defective replication forks is a key step in activation of the S-phase checkpoint.     

Interactions between multiple helminths and the gut microbiota in wild rodents

The gut microbiota is vital to host health and, as such, it is important to elucidate the mechanisms altering its composition and diversity. Intestinal helminths are host immunomodulators and have evolved both temporally and spatially in close association with the gut microbiota, resulting in potential mechanistic interplay. Host–helminth and host–microbiota interactions are comparatively well-examined, unlike microbiota–helminth relationships, which typically focus on experimental infection with a single helminth species in laboratory animals. Here, in addition to a review of the literature on helminth–microbiota interactions, we examined empirically the association between microbiota diversity and composition and natural infection of multiple helminth species in wild mice (Apodemus flavicollis), using 16S rRNA gene catalogues (metataxonomics). In general, helminth presence is linked with high microbiota diversity, which may confer health benefits to the host. Within our wild rodent system variation in the composition and abundance of gut microbial taxa associated with helminths was specific to each helminth species and occurred both up- and downstream of a given helminth''s niche (gut position). The most pronounced helminth–microbiota association was between the presence of tapeworms in the small intestine and increased S24–7 (Bacteroidetes) family in the stomach. Helminths clearly have the potential to alter gut homeostasis. Free-living rodents with a diverse helminth community offer a useful model system that enables both correlative (this study) and manipulative inference to elucidate helminth–microbiota interactions.     

Photochemistry and DNA-affinity of some pyrimidine-substituted styryl-azinium iodides

The relaxation properties of the excited states of three iodides of trans-1,2-diarylethene analogues (where one aryl group is a methylpyridinium, methylquinolinium or dimethylimidazolium group and the other one is a phenyl ring para-substituted by a pyrimidine ring) have been investigated in buffered (pH = 7) aqueous solution. As found in previous works for several analogues, these quaternized salts undergo efficient trans→cis photoisomerization while the yield of the radiative deactivation is very small at room temperature. The solvent effect on the spectral behaviour indicates the occurrence of intramolecular charge transfer which can induce interesting non-linear optical properties. The results of a study of the interactions of these salts with DNA, which might affect the cell metabolism, showed a relatively modest binding affinity for the pyridinium and imidazolium salts and a more substantial affinity for the quinolinium analogue. The formation of ligand-DNA complexes affects only slightly the radiative relaxation yield while leading to a relevant reduction of the isomerization yield. Measurements of the linear dichroism behaviour of the three compounds and comparison with three analogues bearing furan or thienyl groups, which have been found to display different affinity with DNA in previous works, gave interesting information on the nature of the ligand-DNA binding of these compounds.     

A Population-based study of dementia in the oldest old: the Monzino 80-plus Study

Background

Despite being the fastest growing and the most cognitively impaired age group, the oldest olds are under-represented in clinical research. The purpose of this study was to describe the design, methods, and baseline characteristics of the survey population and investigate possible differences in demographic, cognitive, functional, and behavioral characteristics between oldest old with and without any performance on cognitive tests and between oldest old alive and those deceased prior to the interview.

Methods

The Monzino 80-plus Study is a prospective door-to-door population-based survey among 80 years or older residents in the municipalities in the province of Varese, Italy. Dementia cases were identified with a one-phase design. Trained psychologists interviewed both the subject and a proxy informant. The interview included a comprehensive standardized questionnaire together with an array of rating scales and a multidomain cognitive battery to assess cognitive and functional ability, behavioral disturbances and mood.

Results

Information was available for 2,139 of the 2,428 registered individuals aged 80 years or older. Main baseline characteristics of the population are reported and discussed. In comparison with those living, elderly persons who had died before the first visit were older, had twice the rate of institutionalization, poorer cognitive performance and competence, and significantly greater instrumental and basic functional disability. The percentage of elderly persons, alive at baseline, without Mini-Mental State Examination rose rather evenly with age. Moreover, they had significantly worse cognitive competence and functional ability, and reported higher prevalences of depressive symptoms and problem behaviors than those with Mini-Mental State Examination.

Conclusions

Prospective investigation of a large population of oldest old can contribute significantly to understanding the relations between age, cognitive decline, and dementia occurrence. Use of informant-based instruments in surveys in the oldest old is crucial in assessing everyday functioning and changes, especially in participants with no cognitive test performance available. Failure to include information on deceased elderly would underestimate, increasingly with age, the prevalence of cognitive and functional disability in the elderly population.     

Adhesion molecule signalling: not always a sticky business

The signalling activity of cell adhesion molecules (CAMs) such as cadherins, immunoglobulin-like CAMs or integrins has long been considered to be a direct consequence of their adhesive properties. However, there are physiological and pathological processes that reduce or even abrogate the adhesive properties of CAMs, such as cleavage, conformational changes, mutations and shedding. In some cases these 'adhesion deficient' CAMs still retain signalling properties through their cytoplasmic domains and/or their mutated or truncated extracellular domains. The ability of CAMs to activate signal transduction cascades in the absence of cell adhesion significantly extends their range of biological activities.     

The collagen chaperone HSP47 is a new interactor of APP that affects the levels of extracellular beta-amyloid peptides

Alzheimer disease (AD) is a neurodegenerative disorder characterized by progressive decline of cognitive function that represents one of the most dramatic medical challenges for the aging population. Aβ peptides, generated by processing of the Amyloid Precursor Protein (APP), are thought to play a central role in the pathogenesis of AD. However, the network of physical and functional interactions that may affect their production and deposition is still poorly understood. The use of a bioinformatic approach based on human/mouse conserved coexpression allowed us to identify a group of genes that display an expression profile strongly correlated with APP. Among the most prominent candidates, we investigated whether the collagen chaperone HSP47 could be functionally correlated with APP. We found that HSP47 accumulates in amyloid deposits of two different mouse models and of some AD patients, is capable to physically interact with APP and can be relocalized by APP overexpression. Notably, we found that it is possible to reduce the levels of secreted Aβ peptides by reducing the expression of HSP47 or by interfering with its activity via chemical inhibitors. Our data unveil HSP47 as a new functional interactor of APP and imply it as a potential target for preventing the formation and/or growth amyloid plaques.     

Supramaximal exercise mobilizes hematopoietic progenitors and reticulocytes in athletes

Marathon runners show increased circulating CD34+ cell counts and postexercise release of interleukin-6 (IL-6), granulocyte-colony stimulating factor (G-CSF) and flt3-ligand (Bonsignore MR, Morici G, Santoro A, Pegano M, Cascio L, Bonnano A, Abate P, Mirabella F, Profita M, Insalaco G, Gioia M, Vignola AM, Majolino I, Testa U, and Hogg JC. J Appl Physiol 93: 1691-1697, 2002). In the present study we hypothesized that supramaximal ("all-out") exercise may acutely affect circulating progenitors and reticulocytes and investigated possible mechanisms involved. Progenitor release was measured by flow cytometry (n = 20) and clonogenic assays (n = 6) in 20 young competitive rowers (13 M, 7 F, age +/- SD: 17.1 +/- 2.1 yr, peak O2 consumption: 56.5 +/- 11.4 ml.min(-1).kg(-1)) at rest and shortly after 1,000 m "all-out." Release of reticulocytes, cortisol, muscle enzymes, neutrophil elastase, and several cytokines/growth factors was measured. Supramaximal exercise doubled circulating CD34+ cells (rest: 7.6 +/- 3.0, all-out: 16.3 +/- 9.1 cells/mul, P < 0.001), and increased immature reticulocyte fractions; AC133+ cells doubled, suggesting release of angiogenetic precursors. Erythrocyte burst forming units and colony forming units for granulocytes-monocytes and all blood series increased postexercise by 3.4-, 5.5-, and 4.8-fold, respectively (P < 0.01 for all). All-out rowing acutely increased plasma cortisol, neutrophil elastase, flt3-ligand, hepatocyte growth factor, VEGF, and transforming growth factor-beta1, and decreased erythropoietin; K-ligand, stromal-derived factor-1, IL-6, and G-CSF were unchanged. Therefore, all-out exercise is a physiological stimulus for progenitor release in athletes. Release of reticulocytes and proangiogenetic cells and mediators suggests tissue hypoxia as possibly involved in progenitor mobilization.     

Prediction of site-specific amino acid distributions and limits of divergent evolutionary changes in protein sequences

We derive an analytic expression for site-specific stationary distributions of amino acids from the structurally constrained neutral (SCN) model of protein evolution with conservation of folding stability. The stationary distributions that we obtain have a Boltzmann-like shape, and their effective temperature parameter, measuring the limit of divergent evolutionary changes at a given site, can be predicted from a site-specific topological property, the principal eigenvector of the contact matrix of the native conformation of the protein. These analytic results, obtained without free parameters, are compared with simulations of the SCN model and with the site-specific amino acid distributions obtained from the Protein Data Bank. These results also provide new insights into how the topology of a protein fold influences its designability, i.e., the number of sequences compatible with that fold. The dependence of the effective temperature on the principal eigenvector decreases for longer proteins, as a possible consequence of the fact that selection for thermodynamic stability becomes weaker in this case.